1. Field of the Invention
The present invention relates generally to the fields of biophysics and virology. More specifically, the present invention relates to detailed biophysical studies which will lead to both a quantitative understanding of HIV assembly and maturation, and the development of in vitro assays that can be readily adapted for drug screening and evaluation.
2. Description of the Related Art
HIV-1 (and retroviruses in general) assembles through the controlled polymerization of the Gag polyprotein. The Gag polyprotein is transported to the plasma membrane and forms patches within which assembly occurs (1). Transport to the plasma membrane acts to concentrate the Gag protein but is apparently not essential as Gag protein expressed at sufficiently high levels is capable of cytoplasmic assembly (2). During assembly, the patches enlarge and bud outward ultimately pinching off from the cell. During budding, the virion acquires the envelope proteins necessary for receptor binding, as well as a lipid envelope.
Morphologically, the released immature virion presents as an enveloped particle approximately 100 nm in diameter, containing a spherical core (3). The viral protease is incorporated into the virion as part of a Gag-Pol fusion protein, arising from a −1 translational frameshift. Budding activates the protease, which cleaves the Gag polyprotein into the matrix (MA), capsid (CA), and nucleocapsid (NC) domains as well as the “spacer” peptides P2, P1, and the C-terminal P6 domain. The immature virion is metastable, and cleavage of the polyprotein is associated with a profound morphological change in the virion (4). The matrix domain stays associated with the membrane envelope, the capsid domain collapses to form a conical core, and the nucleocapsid domain condenses with the viral RNA in the center of the conical capsid core. The structural rearrangements necessarily arise from the disruption of existing interdomain contacts and the formation of new ones (FIG. 1).
Despite a wealth of structural information about the isolated HIV-1 matrix, capsid, and nucleocapsid domains, little is known about the structure and interactions of these elements within either the immature or mature virus. The structure of the domains themselves may be different in the context of the full length Gag polyprotein than in isolation. The intersubunit interfaces between the domains which promote assembly and their changes upon maturation have not been identified.
Nothing is known of the nature of the large scale motions within the virion required for protease access, maturation, and uncoating. There are two reasons why this is so: the enveloped, non-icosahedral, pleomorphic nature of the virion makes cyrstallographic analysis of the intact virion nearly impossible, and techniques to study the dynamics of large protein complexes have not been readily available.
The prior art is deficient in the lack of detailed quantitative information on the protein/protein interactions mediating HIV assembly and maturation. The present invention fulfills this long-standing need and desire in the art.